Irene Kasindi Meki Irene Kasindi Meki Ki Bum Ahn Ki Bum Ahn William G. Dundon William G. Dundon Tirumala Bharani K. Settypalli Tirumala Bharani K. Settypalli Christoph Leth Christoph Leth Adi Steinrigl Adi Steinrigl Sandra Revilla-Fernández Sandra Revilla-Fernández Friedrich Schmoll Friedrich Schmoll Letizia Ceglie Letizia Ceglie Kouramoudou Berete Kouramoudou Berete Artem Metlin Artem Metlin Madhur Dhingra Madhur Dhingra Norbert Nowotny Norbert Nowotny Giovanni Cattoli Giovanni Cattoli Charles Euloge Lamien Charles Euloge Lamien Springer 2025 Background Recent outbreaks of zoonotic diseases like Ebola, Mpox, dengue fever, and COVID-19 highlight gaps in surveillance and early detection at disease hotspots. Virus family-wide diagnostic assays offer a cost-effective and sensitive alternative to metagenomics for initial virus identification. This study introduces a multiplex family-wide PCR coupled with Nanopore sequencing of amplicons (FP-NSA) for surveillance of novel and known zoonotic respiratory viruses, including influenza A and D viruses (IAV and IDV), alpha (α-), beta (β-), and gamma (γ-) coronaviruses (CoVs). Methods This assay utilized primers in conserved regions of each virus group for multiplex reverse transcription (RT)-PCR coupled with the portable MinION device for rapid Nanopore sequencing. The FP-NSA was optimized using seven IAV subtypes, IDVs, and α- and β-CoVs. The analytical sensitivity of the FP-NSA was assessed using positive controls of known concentrations from each targeted viral family and validated using clinical samples and cell culture isolates from various host species and geographical origins. Potential novel viruses detected in the clinical samples, based on the FP-NSA, were further analyzed using metagenomics sequencing with the Sequence-Independent Single Primer Amplification (SISPA) approach. Results The optimized FP-NSA assay efficiently detected all the targeted viruses singly as well as in co-infection scenarios of multiple respiratory viruses. Evaluation of the assay on 78 selected clinical and cell culture samples (from 184 initially screened) successfully detected IAVs; α-CoVs: porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV) NL63, and HCoV-229E; β-CoVs: HCoV-OC43, severe acute respiratory syndrome (SARS)-CoV-(1), SARS-CoV-2, and MERS-CoV; and γ-CoV infectious bronchitis virus (γ-CoV_IBV) infections. Additionally, the FP-NSA assay discovered a novel γ-CoV_IBV from Guinea that is phylogenetically distant from known genotypes using a SISPA metagenomics approach. Conclusions The assay’s short PCR amplicons enable screening of samples within 4 h, from PCR to sequencing and bioinformatics analysis, providing an adequate number of pathogens’ reads. The portable MinION device makes the assay suitable for pathogen surveillance in disease hotspots and resource-limited regions such as low- and middle-income countries. Thus, the FP-NSA assay is a valuable tool for detecting potential novel and known zoonotic respiratory viruses in the targeted families across various host species. PDFDocument eng 2026-05-11T07:57:42.600539Z 2025 Zoonotic Respiratory Viruses FP-NSA Nanopore Sequencing Multiplex RT-PCR Surveillance 7454359 b application/pdf http://creativecommons.org/licenses/by-nc-nd/4.0/